Botulinum neurotoxins (BoNTs) are the most potent protein toxins known. Given their potent toxicity and their potential use in bio-warfare scenarios, BoNTs are included among the Category A select agents. The currently available pentavalent toxoid vaccine requires multiple boosters before being effective, and protects unequally against the different BoNT serotypes. There is a critical need for BoNT vaccines that will induce rapid protection in situations where the threat of BoNT exposure is imminent as well as to provide effective immune responses to each constituent of a multivalent vaccine. To achieve rapid and more robust immune responses to Hc we have pursued a strategy of linking Hc antigens to R4, a novel adjuvant developed by us that efficiently loads vaccine antigens onto dendritic cells (DCs). By specifically targeting the Hc antigen to DCs, the critical early steps in the immune response are hastened. We have expressed BoNT/A Hc as a fusion protein with R4 to produce HcR4 and have demonstrated that HcR4 induces more rapid and robust immune responses to BoNT/A Hc antigen than can be achieved using the Hc antigen alone. Based on these results we hypothesize that an effective rapid- acting multivalent vaccine against BoNTs can be developed using HcR4 fusion proteins. The goal of this project is to advance the development of a new class of BoNT vaccines that represent a significant improvement over current vaccine technologies. The proposed studies will achieve these goals through development of HcR4 ligands for BoNT serotypes B and E for use in combination with the already produced and characterized BoNT/A HcR4 as a multivalent vaccine. We will test whether this multivalent vaccine can generate rapid and protective immune responses to BoNTs when administered as a single or multidose parenteral vaccine. In addition, the multivalent BoNT vaccine will be tested as a mucosal vaccine for the generation of systemic and mucosal immune responses to BoNT. This project will achieve the next milestones in the development of the HcR4 ligands: 1) We will develop HcR4 immunogens for BoNT serotypes B and E for use in combination with the already developed and characterized BoNT/A HcR4. 2) We will establish the efficacy of a multivalent HcR4 vaccine as a rapid- protection, one-shot vaccine, and as a multi-dose vaccine, and; 3) Determine if mucosal/nasal administration of HcR4 can result in the rapid induction of systemic and mucosal antibody responses against BoNTs. These studies are intended to deliver sufficient proof-of-principle for advancing the polyvalent BoNT vaccine into clinical development. The know-how gained in the current studies will be useful in the future for development of novel and improved vaccines to other toxins and infectious agents.